Fig 2: Mutations in the major SUMOylation acceptor sites of a-syn exacerbate METH-induced a-syn aggregation in vivo. Adenovirus were injected into the right striatum of mice using a standard stereotaxic positioning system. After 2 days of recovery, mice received saline or METH by i.p. injection. Striatal tissues (right side) were harvested at 24 h after the injection of the last dose. The tissues were immunoprecipitated with an anti-SUMO-1 antibody, followed by western blot with an anti-a-syn antibody. Tissues immunoprecipitated with IgG were used as a negative control. Western blot (A,C) and quantitative analyses (A1,C1) were performed to evaluate the efficiency of WT a-syn and a-syn-KR expression. Immunofluorescence staining of mouse striatum sections showed that a-syn expression increased after injection of adenovirus (B). Western blot (D–F) and quantitative analyses (D1,E1–5,F1–6) were also performed to the levels of the SUMO-1, UbE1, Ub, LC3-II, P-62 and lysosomal associated membrane protein 2A (LAMP2A) proteins and the expression of SUMOylated a-syn. ß-Actin was used as a loading control. *p < 0.05 compared with the AAV2-NC or control group. **p < 0.05 compared with the AAV2-a-syn group. #p < 0.05 compared with the AAV2-a-syn + METH-treated group. The data shown in (A,C,D,F) were analyzed using one-way ANOVA followed by LSD post hoc analyses, whereas the data shown in (E) were analyzed using the Mann-Whitney U test.

Fig 3: Model for the formation of ATR-signalling bodies.Upon addition of a gapped DNA substrate in human cell-free extracts, poly(ADP-ribosyl)ation accelerates the recruitment of DNA damage signalling proteins, including the ubiquitin-activating enzyme UBA1. The coupling of pADPr-seeded protein assembly with protein ubiquitylation and protein phosphorylation prevents the formation of nonfunctional protein aggregates and promotes dedicated protein–protein interactions required for ATR signalling.

Fig 4: UBA1 residues conserved in animals (but not in fungi) are critical for pADPr binding.(A) Sequence logos of the alignment of the region shown in Fig 5B. The height of the letters in each position represents the degree of sequence conservation. Colours are used for differentiation among hydrophilic (blue), hydrophobic (black), or neutral amino acids (green). Error bars correspond to the Bayesian 95% confidence interval of the height. Arrows indicate the positions of the amino acids chosen for mutagenesis. These seven amino acids are not conserved between Animalia and Fungi. (B) Increasing amounts (5 to 80 pM) of purified MBPUBA1(571–800) fragments containing the indicated amino acid substitutions were spotted on a nitrocellulose membrane, incubated with purified pADPr chains, and washed. The retention of pADPr was analysed using an anti-pADPr antibody. UBA1 protein fragments used in the pADPr binding assay were revealed using an anti-MBP antibody (middle panel) or resolved by PAGE and stained with Coomassie Brilliant Blue (bottom panel). (C) U2OS cells were transfected with pAIO vector encoding an anti-UBA1 shRNA and an shRNA-resistant UBA1 cDNA encoding wild-type or mutated UBA1, as indicated, and treated with doxycyclin for 3 d. Cells were exposed to 1 µM CPT for increasing amounts of time (as indicated). Indicated proteins were detected by Western blotting.

Fig 5: UBA1 is recruited to an ATR-activating structure and is associated with ongoing replication forks.(A) Experimental scheme: Biotinylated duplex DNA structures are incubated with nuclear extract in the presence of ATP. After 10 min at 37°C, DNA-bound proteins are pulled down using streptavidin-coated beads. To monitor protein ubiquitylation in vitro, reaction mixtures are supplemented with recombinant FLAG-ubiquitin. (B) Nuclear extracts were incubated with the indicated biotinylated DNA substrates. DNA-bound proteins were isolated, resolved by PAGE, stained with silver, and identified by mass spectrometry. The most abundant proteins are indicated. (C) Indicated proteins were pulled down with streptavidin-coated beads coupled to duplex DNA or gapped duplex DNA and detected by Western blotting. The duplex DNA and gapped duplex DNA substrates are represented schematically. Biotin (black circles) and streptavidin-coated beads (dented grey circles) are shown. (D) Indicated proteins were isolated by iPOND and detected by Western blotting. HEK293 cells were pulse-labelled with 5-ethynyl-2'-deoxyuridine (EdU) for 10 min and chased with thymidine for 60 min. In no click samples (no Clk), desthiobiotin-TEG azide was replaced by DMSO. (E) Bar plot showing average peptides intensities (MaxQuant label-free quantification) corresponding to the indicated proteins. HeLa S3 cells were pulse-labelled with EdU for 5 min and chased with thymidine for 120 min. Pulse experiment has been repeated three times and chase experiment two times. Error bars represent the standard variation. The UBA1 peptides identified are listed in Table S1.